High throughput screens (“HTS”) and high content screens (“HCS”) are essential techniques for use in drug discovery and related areas. Standard HTS use mixtures of compounds and biological reagents along with some indicator compound loaded into arrays of wells in standard microtiter plates with 96 or 384 wells. The signal measured from each well, either fluorescence emission, optical density, or radioactivity, integrates the signal from all the material in the well giving an overall population average of all the molecules in the well. In contrast to HTS, various HCS have been developed to address the need for more detailed information about the temporal-spatial dynamics of cell constituents and processes. HCS automate the extraction of multicolor fluorescence information derived from specific fluorescence-based reagents incorporated into cells (Giuliano and Taylor (1995), Curr. Op. Cell Biol. 7:4; Giuliano et al. (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405). Cells are analyzed using an optical system that can measure spatial, as well as temporal dynamics. (Farkas et al. (1993) Ann. Rev. Physiol. 55:785; Giuliano et al. (1990) In Optical Microscopy for Biology. B. Herman and K. Jacobson (eds.), pp. 543-557. Wiley-Liss, New York; Hahn et al (1992) Nature 359:736; Waggoner et al. (1996) Hum. Pathol. 27:494). The concept is to treat each cell as an object that has spatial and temporal information on the activities of the labeled constituents.
Image acquisition is one of the key procedures in the feature extraction process utilized in HTS and HCS. In order to automate the analysis of cells spread in space, it is necessary to automate the process of image acquisition from different parts of a slide or different wells or fields within a well of a microtiter plate. Prior autofocus methods involve computation of some image statistic for all different focal planes, and then selection of the focal plane with the highest statistic. This method is costly both in terms of time and in terms of photobleaching the sample and limits the efficiency of HT and HC screening. Furthermore, problems can appear due to irregularities in the slide or microtiter plate such that the position of the plate relative to the objective is different at different locations on the slide or microtiter plate.
Thus, there exists a need in the art for a more rapid autofocusing method to improve the efficiency and throughput of HTS and HCS, and one that corrects for irregularities in the sample plate. The present invention fulfills this need in the art.